CN117887709A - 一种随机改变基因dna序列的基因杂交方法 - Google Patents
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Abstract
本发明属于基因工程技术领域,具体为一种随机改变基因DNA序列的基因杂交方法。本发明将不同来源且具有高同源性的基因DNA序列随机拼接进行杂交,得到大量不同于原始序列的全新序列,再将全新序列构建至载体上进行表达后筛选,得到更高效率的酶。具体包括在基因库中找出不同物种中相同功能的氨基酸序列,通过计算机辅助选出具有高度同源性的氨基酸序列进行分析后优化密码子;以优化的DNA序列为模板进行设计,在每条模板上连续拉出多条寡核苷酸序列,把每条模板都分割成为多条寡核苷酸,分别合成每条寡核苷酸;进行两轮PCR,将产物进行分子克隆转化进入大肠杆菌中,获得大量含有全新基因序列的转化子,挑出所有转化子后进行测序。
Description
技术领域
本发明属于基因工程技术领域,具体涉及随机改变基因序列的基因杂交方法。
背景技术
D-阿洛酮糖是D-果糖的C-3差向异构体,是稀有糖家族的重要成员。D-阿洛酮糖属于己酮糖,是一种六碳糖,具有较高的甜度和较低的能量,有很多生理保健功能[1],能够减少脂肪堆积、降低餐后血糖水平、清除活性氧簇、治疗动脉粥样硬化疾病、增强胰岛素抗性和保护神经等[2-4]。D-阿洛酮糖在自然界极度稀缺,化学合成法可以制备D-阿洛酮糖,但这些方法存在经济性差、环境污染严重等问题[5]。与化学法相比,生物转化通常单步反应即可获得目的产物,反应条件温和、活性高、使用剂量低,无需有毒试剂,环境相容性好[5]。
D-阿洛酮糖3-差向异构酶(DPEase)催化D-果糖和D-阿洛酮糖在C-3位置发生可逆的差向异构化反应[6],可以有效地催化D-果糖和D-阿洛酮糖的相互转化[7],在D-阿洛酮糖生物转化中起着至关重要的作用。酮糖3-差向异构酶的基因被推测分布于很多种微生物中[8],不同来源的酮糖3-差向异构酶,催化效率各不相同,酶学性质也有较大的差别[9]。通过基因工程进行酶分子改造是改变D-阿洛酮糖3-差向异构酶酶学性质的重要手段之一[10]。
在基因工程中,定点突变是产生氨基酸序列改变的突变体的重要工具[11],通过改变酶的氨基酸序列进而改善酶的性质,如催化活性、热稳定性和耐化学性[12]。用于酶研究、蛋白质结构和功能之间关系的研究以及基因或其调控序列的功能分析[13-15]。传统的PCR技术一次只能产生一个基因,有很大的局限性。本发明通过两步PCR将不同来源的同一个基因进行随机拼接,每次PCR都能产生多种序列不同的基因,能够得到各种各样的DNA序列,从而产生大量不同的DPEase,能够快速建立DPEase突变体库。
发明内容
本发明的目的在于提出一种随机改变基因DNA序列的基因杂交方法,以解决上述背景技术中遇到的问题。
本发明提供的随机改变基因DNA序列的杂交方法,是将不同来源且具有高同源性的相同功能基因的DNA序列随机拼接进行杂交后,得到大量不同于原始序列的全新的序列,再将得到的全新序列构建至载体上进行表达后筛选,得到更高效率的酶,具体步骤为:
(1)在基因库中找出不同物种中相同功能的氨基酸序列,通过计算机辅助选出具有高度同源性的氨基酸序列进行分析后优化密码子;
(2)然后分别在每条优化之后的DNA序列两端加上相同的上游引物和下游引物,以优化之后的DNA序列为模板进行设计,在每条模板上连续拉出多条寡核苷酸序列,这样把每条模板都分割成为多条寡核苷酸,分别合成每条寡核苷酸;
(3)分别取所有寡核苷酸混合,使用高保真Taq酶进行第一轮PCR,在第一轮PCR中,不同来源的寡核苷酸片段之间由于有较高的同源性随机拼接起来生成全新的模板;以第一轮PCR产物为模板,以两端相同的序列为引物进行第二轮PCR,将第二轮PCR产物进行分子克隆转化进入细菌感受态中,涂布平板,从而获得大量含有全新基因序列的转化子,挑出所有的转化子后进行测序。
进一步地,具体操作流程为:
S1、从数据库中搜索不同来源且高度同源的基因DNA序列的氨基酸序列,利用网站对这些氨基酸序列进行密码子优化;
S2、以已经优化的DNA序列为模板,使用生物学软件分别在每条DNA模板上连续拉出多条寡核苷酸序列(寡核苷酸的条数需为偶数条),将每条寡核苷酸分别合成;
S2、使用ddH2O将寡核苷酸稀释为100mM/L;
S3、取所有寡核苷酸在EP管中,混匀,得到混合寡核苷酸;
步骤S4、使用高保真Taq酶,加入混合寡核苷酸进行第一轮PCR,本轮PCR中,由于DNA序列的同源性,不同模板拉出的寡核苷酸序列随机重新拼接,生成全新的模板;
S5、在DNA序列的基因杂交中,加到基因DNA序列(例如DPEase基因)两端的上下游引物,以第一轮PCR产物为模板,使用高保真Taq酶进行第二轮PCR,大量扩增第一轮PCR生成的模板;
S6、不同来源的基因DNA序列大小相近,将第二轮PCR产物跑琼脂糖凝胶电泳,割胶回收与模板DNA大小相近的DNA条带;
S7、将纯化后的DNA片段与质粒采用T4 DNA连接酶进行连接,并转化到细菌感受态中;
S8、长出的单克隆通过菌落PCR进行验证,对电泳后条带大小正确的克隆摇菌后抽提质粒,并进行测序确定其具体序列信息;本步骤中可一次获得多个不同序列的单克隆;
S9、对阳性单克隆分别进行酶促反应,并除盐后进行HPLC检查,确定阳性克隆的转化率。
本发明提供了一种改变基因序列的基因杂交方法,只需一次构建就能获得多个表达不同效率酶的杂交菌;所使用的克隆引物是通过软件设计后优选出来的;连接所用的DNA片段是对两轮PCR后获得的产物进行回收纯化获得的;构建的克隆是连接到质粒后进行转化,可一次获得大量单克隆;所获得的杂交菌可经测序后获得具体序列信息;所获得的杂交菌可通过HPLC检测转化率,筛选出转化率最高的菌株,节省时间的同时大大提高了工作效率。
附图说明
图1为本发明获得的几种不同克隆基因序列比对图。其中,优化模板为选出的不同物种来源的基因通过计算机进行密码子优化后得到的DNA序列,在序列比对图中,蓝色为杂交菌与优化模板相比相同的碱基,空白为杂交菌与优化模板相比发生了碱基缺失、增加或突变;本附图中a.Hb-24与优化模板序列比对结果;b.Hb-106与优化模板序列比对结果;c.Hb-109与优化模板序列比对结果;d.Hb-120与优化模板序列比对结果;e.Hb-130与优化模板序列比对结果;f.Hb-300与优化模板序列比对结果。
图2为基因杂交成功的序列和优化后的序列比对图。其中,优化序列为在网站中对选出的氨基酸序列进行密码子优化后的DNA序列,每条箭头代表一条序列。蓝色部分为同源序列,空白部分为DNA碱基缺失、增加或突变。
图3为本发明技术流程示意图。
图4为不同基因杂交细菌将D-果糖转化为D-阿洛酮糖的转化率柱状图。横坐标为基因杂交细菌的编号,纵坐标为基因杂交细菌将D-果糖转化为D-阿洛酮糖的转化率不同的基因杂交细菌将D-果糖转化为D-阿洛酮糖的转化率不同。
具体实施方式
下面以D-阿洛酮糖3-差向异构酶(DPEase)基因为例,进一步介绍本发明。
步骤S1、从数据库中搜索不同来源且高度同源的DPEase基因的几条氨基酸序列,利用网站对这些氨基酸序列进行密码子优化。
步骤S2、以已经优化的DNA序列为模板,使用生物学软件分别在每条DNA模板上连续拉出多条长度在60bp以内的寡核苷酸序列(寡核苷酸的序列需为偶数条,DPEase基因的长度约900bp,每条模板共设计出32条寡核苷酸序列),将每条寡核苷酸分别合成;
步骤S2、使用ddH2O将寡核苷酸稀释为100mM/L;
步骤S3、取所有寡核苷酸各1μl在EP管中,混匀,得到混合寡核苷酸;
步骤S4、使用高保真Taq酶,加入混合寡核苷酸进行第一轮PCR,本轮PCR中,由于DNA序列的同源性,不同模板拉出的寡核苷酸序列随机重新拼接,生成全新的模板;
步骤S5、在DPEase基因的基因杂交中,加到DPEase基因两端的上下游引物分别命名为:DPE-001,DPE-002,以第一轮PCR产物为模板,DPE-001,DPE-002为上下游引物,使用高保真Taq酶进行第二轮PCR,大量扩增第一轮PCR生成的模板;
步骤S6、不同来源的DPEase基因大小约为900bp,将第二轮PCR产物跑琼脂糖凝胶电泳,割胶回收大小在900bp左右的DNA条带;
步骤S7、将纯化后的DNA片段与质粒进行连接,并转化到大肠杆菌BL21(DE3)感受态中;
步骤S8、长出的单克隆通过菌落PCR进行验证,对电泳后条带大小正确的克隆摇菌后抽提质粒,并进行测序确定其具体序列信息;
步骤S9、对阳性单克隆分别进行酶促反应,并除盐后进行HPLC检查,确定阳性克隆的转化率。
结果:如图1所示为部分成功基因杂交的基因序列和原始的模板序列比对图,图4为含有不同的杂交基因的细菌将D-果糖转化为D-阿洛酮糖的转化率不同,有些杂交基因转化率为0,有些杂交基因的转化率高达26.6%。
参考文献
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[3]Suna,S.,Yamaguchi,F.,Kimura,S.,Tokuda,M.,Jitsunari,F.,2007.Preventive effect of D-psicose,one of rare ketohexoses,on di-(2-ethylhexyl)phthalate(DEHP)-induced testicular injury in rat.Toxicol.Lett.173,107-117.
[4]Takata,M.K.,Yamaguchi,F.,Nakanose,K.,Watanabe,Y.,Hatano,N.,Tsukamoto,I.,Nagata,M.,Izumori,K.,Tokuda,M.,2005.Neuroprotective effect of D-Psicose on6-hydroxy dopamine-induced apoptosis in rat pheochromocytoma(PC12)cells.J.Biosci.Bioeng.100,511–516.
[5]贾东旭,孙晨奕,彭晨等.D-阿洛酮糖及其合成研究进展[J].食品与发酵工业,2021,47(03):211-217.
[6]Kim H J,Hyun E K,Kim Y S,et al.Characterization of anAgrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose toD-psicose[J].Applied and Environmental Microbiology,2006,72(2):981-985.
[7]李秋喜.D-阿洛酮糖3-差向异构酶的固定化技术研究[D].江南大学,2014.
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[9]张文立.D-阿洛酮糖3-差向异构酶的高效表达、酶学性质及分子改造[D].江南大学,2018.
[10]Choi J G,Ju Y H,Yeom S J,et al.Improvement in the thermostabilityof D-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis[J].Applied and Environmental Microbiology,2011,77(20):7316-7320.
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[12]Combined Overlap Extension PCR Method for Improved Site DirectedMutagenesis
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Claims (2)
1.一种随机改变基因DNA序列的杂交方法,其特征在于,具体步骤为:
是将不同来源且具有高同源性的相同功能基因的DNA序列随机拼接进行杂交后,得到大量不同于原始序列的全新的序列,再将得到的全新序列构建至载体上进行表达后筛选,得到更高效率的酶,具体步骤为:
(1)在基因库中找出不同物种中相同功能的氨基酸序列,通过计算机辅助选出具有高度同源性的氨基酸序列进行分析后优化密码子;
(2)然后分别在每条优化之后的DNA序列两端加上相同的上游引物和下游引物,以优化之后的DNA序列为模板进行设计,在每条模板上连续拉出多条寡核苷酸序列,每条寡核苷酸序列长度在60bp以内,这样把每条模板都分割成为多条寡核苷酸,分别合成每条寡核苷酸;
(3)取所有寡核苷酸各1μl混合,使用高保真Taq酶进行第一轮PCR,在第一轮PCR中,不同来源的寡核苷酸片段之间由于有较高的同源性随机拼接起来生成全新的模板;以第一轮PCR产物为模板,以两端相同的序列为引物进行第二轮PCR,将第二轮PCR产物进行分子克隆转化进入大肠杆菌中,从而获得大量含有全新基因序列的转化子,挑出所有的转化子后进行测序。
2.根据权利要求1所述的机改变基因DNA序列的杂交方法,其特征在于,具体操作流程为:
S1、从数据库中搜索不同来源且高度同源的基因DNA序列的氨基酸序列,利用网站对这些氨基酸序列进行密码子优化;
S2、以已经优化的DNA序列为模板,使用生物学软件分别在每条DNA模板上连续拉出多条寡核苷酸序列,将每条寡核苷酸分别合成;
S2、使用ddH2O稀释寡核苷酸;
S3、取所有寡核苷酸在EP管中,混匀,得到混合寡核苷酸;
S4、使用高保真Taq酶,加入混合寡核苷酸进行第一轮PCR,本轮PCR中,由于DNA序列的同源性,不同模板拉出的寡核苷酸序列随机重新拼接,生成全新的模板;
S5、在DNA序列的基因杂交中,加到基因DNA序列两端的上下游引物分别命名为:DPE-001,DPE-002,以第一轮PCR产物为模板,DPE-001,DPE-002为上下游引物,使用高保真Taq酶进行第二轮PCR,大量扩增第一轮PCR生成的模板;
S6、不同来源的基因DNA序列大小相近,将第二轮PCR产物跑琼脂糖凝胶电泳,割胶回收与模板DNA大小相近的DNA条带;
S7、将纯化后的DNA片段与质粒采用T4 DNA连接酶进行连接,并转化到大肠杆菌感受态中;
S8、长出的单克隆通过菌落PCR进行验证,对电泳后条带大小正确的克隆摇菌后抽提质粒,并进行测序确定其具体序列信息;
S9、对阳性单克隆分别进行酶促反应,并除盐后进行HPLC检查,确定阳性克隆的转化率。
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